Gel Microbeads Research Articles

High-Throughput Gel Microbeads as Incubators for Bacterial Competition Study.

Bacteria primarily live in structured environments, such as colonies and biofilms, attached to surfaces or growing within soft tissues. They are engaged in local competitive and cooperative interactions impacting our health and well-being, for example, by affecting population-level drug resistance. Our knowledge of bacterial competition and cooperation within soft matrices is incomplete, partly because we lack high-throughput tools to quantitatively study their interactions. Here, we introduce a method to generate a large amount of agarose microbeads that mimic the natural culture conditions experienced by bacteria to co-encapsulate two strains of fluorescence-labeled Escherichia coli. Focusing specifically on low bacterial inoculum (1-100 cells/capsule), we demonstrate a study on the formation of colonies of both strains within these 3D scaffolds and follow their growth kinetics and interaction using fluorescence microscopy in highly replicated experiments. We confirmed that the average final colony size is inversely proportional to the inoculum size in this semi-solid environment as a result of limited available resources. Furthermore, the colony shape and fluorescence intensity per colony are distinctly different in monoculture and co-culture. The experimental observations in mono- and co-culture are compared with predictions from a simple growth model. We suggest that our high throughput and small footprint microbead system is an excellent platform for future investigation of competitive and cooperative interactions in bacterial communities under diverse conditions, including antibiotics stress.

N-doped highly microporous carbon derived from the self-assembled lignin/chitosan composites beads for selective CO2 capture and efficient p-nitrophenol adsorption

Green and low-cost biomass-based porous carbon adsorbents have a good prospect for the removal of some environmental pollutants. Here, sodium lignosulphonate, chitosan, and activator were firstly combined into wet gel microbeads through in situ self-assembly method, and then derived the novel porous carbon adsorption materials (LC-Cs) by freeze-drying and one-step activation carbonization. The results showed that LC-Cs had large specific surface areas (892.4∼1307.8 m2/g), high microporosity (87.4∼89.2%), and rich nitrogen content (3.01∼4.92%), and these structural parameters showed good linear dependence with the dosage ratio of lignin to chitosan. Next, LC-Cs carbon showed efficient CO2 capture (173.9–215.2 mg/g) and high CO2/N2 selectivity (up to IAST:145.9, Henry’s law: 23.5), specially, the relationships between the CO2 capture and the structural parameters were investigated in detail, and also performed DFT calculation. In addition, LC-C22 had the maximum adsorption capacity of p-nitrophenol (PNP) as 592.8 mg/g, and the adsorption isotherms and kinetic study indicated that they belonged to the multilayer adsorption on the heterogeneous surfaces, and the chemical adsorption played the dominated roles, and then further studied the adsorption mechanism by characterization techniques. This work provided a green and facile strategy for preparing structurally controllable N-doped porous carbon materials, as well as offered good potential adsorbents for PNP removal and CO2 capture.

Thermally-modulated shape transition at the interface of soft gel filament and hydrophobic substrate

A liquid filament may pinch off into different shapes on interacting with a soft surface, as modulated by the interplay of inertial, capillary, and viscous forces. While similar shape transitions may intuitively be realized for more complex materials such as soft gel filaments as well, their intricate controllability towards deriving precise and stable morphological features remains challenging, as attributed to the complexities stemming from the underlying interfacial interactions over the relevant length and time scales during the sol–gel transition process. Circumventing these deficits in the reported literature, here we report a new means of precisely-controlled fabrication of gel microbeads via exploiting thermally-modulated instabilities of a soft filament atop a hydrophobic substrate. Our experiments reveal that abrupt morphological transitions of the gel material set in at a threshold temperature, resulting in spontaneous capillary thinning and filament breakup. We show that this phenomenon may be precisely modulated by an alteration in the hydration state of the gel material that may be preferentially dictated by its intrinsic glycerol content. Our results demonstrate that the consequent morphological transitions give rise to topologically-selective microbeads as an exclusive signature of the interfacial interactions of the gel material with the deformable hydrophobic interface underneath. Thus, intricate control may be imposed on the spatio-temporal evolution of the deforming gel, facilitating the inception of highly ordered structures of specific shapes and dimensionalities on demand. This is likely to advance the strategies of long shelf-life analytical biomaterial encapsulations via realizing one-step physical immobilization of bio-analytes on the bead surfaces as a new route to controlled materials processing, without demanding any resourced microfabrication facility or delicate consumable materials.

Biodegradable Crosslinked Chitosan Gel Microbeads with Controlled Size, Prepared by Membrane Emulsification-External Gelation and Their Application as Reusable Adsorption Materials

Gel microbeads composed of chitosan biopolymers were synthesized via membrane emulsification, as novel adsorbents for the adsorption of anionic azo dye (Acid Orange 7 (AO7)) and protein (bovine serum albumin (BSA)). The synthesis route involved the production of size-controlled water-in-oil emulsion droplets containing the chitosan solution using a porous, hydrophobic polytetrafluoroethylene membrane. The resulting size-controlled emulsion droplets were successfully converted into stable gel microbeads via external gelation, followed by chemical crosslinking using ethylene glycol diglycidyl ether (EGDE). Scanning electron microscopy results revealed the size of the gel microbeads having a diameter ranging from 15 to 20 µm, which was comparable to the size of the emulsion droplets. The effect of the crosslinking reaction between the prepared gel microbeads with EGDE was confirmed via Fourier transform infrared spectroscopy. The batch adsorption studies employed the Langmuir adsorption model with maximum adsorption capacities at pH values of 5 and 6 for AO7 dye (qmax=1.84 g/g) and BSA (qmax=0.87 g/g), respectively, with electrostatic interaction being the dominant mechanism of adsorption for both the azo dye and protein. The prepared crosslinked chitosan gel microbeads in this study exhibited excellent recyclability up to 30 times without a decline in their adsorptive capacities, a shelf life of >100 d, and a fair susceptibility toward enzymatic biodegradation, making them some of the most effective and versatile green adsorbents for wastewater treatment.

Morphological Manipulation of DNA Gel Microbeads with Biomolecular Stimuli.

Hydrogels are essential in many fields ranging from tissue engineering and drug delivery to food sciences or cosmetics. Hydrogels that respond to specific biomolecular stimuli such as DNA, mRNA, miRNA and small molecules are highly desirable from the perspective of medical applications, however interfacing classical hydrogels with nucleic acids is still challenging. Here were demonstrate the generation of microbeads of DNA hydrogels with droplet microfluidic, and their morphological actuation with DNA strands. Using strand displacement and the specificity of DNA base pairing, we selectively dissolved gel beads, and reversibly changed their size on-the-fly with controlled swelling and shrinking. Lastly, we performed a complex computing primitive—A Winner-Takes-All competition between two populations of gel beads. Overall, these results show that strand responsive DNA gels have tantalizing potentials to enhance and expand traditional hydrogels, in particular for applications in sequencing and drug delivery.

Bed expansion properties of tissue engineering particles in a fluidised bed bioreactor

Fluidised bed bioreactors (FBBs) are a promising alternative to provide high cell densities and a more defined culture environment for 2D and 3D cell cultures. However, for their wider use in biotechnology applications, a better understanding of their hydrodynamics is required. In this work, the fluidisation properties of three common particles in tissue engineering - single cells, cells clusters, and gel microbeads - were evaluated and compared to established liquid fluidisation correlations. Only a few studies to date have attempted to characterise the hydrodynamic behaviour of gel beads, while none were found for the other two particle types studied here. All particles were in the micrometre size range and their densities were found to be near-equal to the fluid phase. The particle terminal velocities were measured and corrected for hindrance effects, but these values were poorly predicted by equations proposed for rigid smooth spheres. Bed expansion data was presented in the form of logarithmic plots of velocity against voidage and analysed in terms of Richardson & Zaki parameters (n, expansion index and k, correction factor). Linear relationships were observed in these graphs; however, the fitting parameters were incorrectly predicted by the established equations. Therefore, modified Richardson-Zaki correlations were developed by using experimental parameters for n and k, to enable other users to set up FBBs for tissue engineering applications.

Self-organization of hepatocyte morphogenesis depending on the size of collagen microbeads relative to hepatocytes

Recent advances in microfabrication technologies have enabled us to construct collagen gel microbeads, which can be cultured with hepatocytes. However, little is known about the hepatocyte–collagen gel microbead interactions. Here, we aimed to clarify the effects of the balance between cell–cell and cell–collagen gel microbead interactions on hepatocyte morphogenesis and functions. The magnitude of cell–microbead interactions was controlled by changing the size of the microbeads, which were smaller than, comparable to, and larger than hepatocytes. These small, medium, and large microbeads were cultured separately with primary hepatocytes. Phase-contrast and time-lapse imaging revealed that the medium microbeads significantly induced the construction of 3D structures composed of the microbeads and hepatocytes in a self-organizing manner, whereas hepatocytes formed 2D monolayers with the small or large microbeads. These results suggest that only the medium microbeads induced the 3D tissue formation of hepatocytes. Furthermore, liver-specific functions, such as albumin secretion and ammonia clearance, were significantly upregulated in the 3D structures. These findings are critical to understand how to control the construction of 3D hepatocyte tissues with hydrogel microbeads in the context of biofabrication.

Novel sequential separation and determination of a quaternary mixture of fungicides by using an automatic fluorimetric optosensor

ABSTRACTA versatile flow-through multi-optosensor is proposed for the separation and spectrofluorimetric determination of mixtures of four widely used pesticides: carbendazim, thiabendazole, carbaryl and o-phenylphenol at µg g−1 levels in fruits. The flow system is based on the online pre-concentration and separation of the pesticides on a solid sensing microzone, followed by the sequential measurement of their native fluorescence. The separation of the pesticides takes place on a solid support located in the same flow cell, on which analytes are temporarily immobilized and separated from the matrix due to their different retention/desorption kinetics when they interact with the C18 silica gel microbeads. Suitable analytical parameters were obtained for the selected analytes, with method detection and quantification limits ranging between 0.1–0.5 and 0.2–1.6 µg g−1, respectively. These values comply with the maximum residue limits (MRLs) established by the Codex Alimentarius for these commodities; in addition, carbendazim, thiabendazole and ortho-phenylphenol comply with the MRLs of The European Union. The developed method was applied to the analysis of citrus fruits by performing recovery studies. Recoveries between 85% and 115% were obtained in all cases, and the results were confirmed by a liquid chromatography–mass spectrometry reference method.

The impact of target stiffness on NK cell activation

Abstract A diverse number of cell functions and behaviour are regulated by the mechanical properties of surrounding tissue. In addition, mechanical forces have been shown to impact synapse formation and signalling by T and B cells. Here, we set out to establish whether or not target cell tension impacts NK cell responses. To address this, human primary NK cells were placed on polyacrylamide substrates of varying rigidity coated with activating antibodies against NKp30 and LFA-1. NK cell activation measured by IFNγ secretion and degranulation were reduced on softer substrates. To better model the 3D interaction between NK cells and target cells, we synthesised sodium alginate cell-sized microbeads crosslinked with calcium chloride, where the inherent stiffness of the beads was controlled to be low (2-15kPa), medium (25-50kPa), or high stiffness (75-400kPa). Beads were coated with increasing concentrations of antibody to create surrogate targets with specific ligands and controlled stiffness. NK cells conjugated to stiff beads coated with mAb against NKp30, showed greater actin clustering, as well as perforin and MTOC polarisation towards the bead, to a far greater extent than those conjugated to softer beads. Thus, gel microbeads can be used to characterise the mechanosensitivity of immune synapses in 3D, and establish a role for target stiffness in NK cell activation.

Human Cell Encapsulation in Gel Microbeads with Cosynthesized Concentric Nanoporous Solid Shells

AbstractEncapsulation of therapeutic cells in core–shell microparticles has great promise for the treatment of a range of health conditions. Unresolved challenges related to control of the particle morphology, mechanical stability, and immunogenicity hinder dissemination of this promising approach. Here, a novel polymer material for cell encapsulation and a combined novel, easy to control, synthesis method are introduced. Core–shell cell encapsulation is demonstrated with a concentric core–shell morphology formed during a single UV exposure, resulting in particles that consist of a synthetic hydrogel core of polyethylene glycol diacrylate and a solid, but porous, shell of off‐stoichiometric thiol‐ene. The encapsulated human cells in 100 µm diameter particles have >90% viability. The average shell thickness is controlled between 7 and 13 µm by varying the UV exposure, and the shell is measured to be permeable to low molecular weight species (<180 Da) but impermeable to higher molecular weight species (>480 Da). The unique material properties and the orthogonal control of the microparticle core size, shell thickness, shell permeability, and shell surface properties address the key unresolved challenges in the field, and are expected to enable faster translation of novel cell therapy concepts from research to clinical practice.

Cross-linkable chitosan-based hydrogel microbeads with pH-responsive adsorption properties for organic dyes prepared using size-tunable microchannel emulsification technique

Cross-linked chitosan hydrogel microbeads were prepared from monodisperse water-in-oil (W/O) emulsions and their adsorption properties for an anionic organic dye, Acid Orange 7 (AO7), were investigated. Preparation of monodisperse W/O emulsions with a mean diameter of 22–28μm and coefficient of variation below 10% were achieved by microchannel (MC) emulsification. The mean diameter and diameter distribution were affected by the viscosity and flow rate of the chitosan solution pressed into MCs. Chitosan gel microbeads were formed by gelation of chitosan-containing water droplets by alkali treatment followed by cross-linking treatment. The resulting microbeads still had a narrow diameter distribution. The pH-responsive adsorption property of chitosan gel microbeads was studied using AO7. The experiment results indicated that negatively charged AO7 molecules adsorbed onto positively charged chitosan gel microbeads via electrostatic interaction. The chitosan gel microbeads were stable for more than 100days and could be reused in repetitive adsorption-desorption cycles.

Formulation of Uniform-sized Agar Gel Microbeads from Water-in-oil Emulsion Prepared Using Microchannel Emulsification under Controlled Temperature

Formulation of Uniform-sized Agar Gel Microbeads from Water-in-oil Emulsion Prepared Using Microchannel Emulsification under Controlled Temperature

Characterization and stability analysis of biopolymeric matrices designed for phage-controlled release.

Alginate and low methoxylated pectin gel matrices emulsified with oleic acid were studied for phage oral delivery. Matrix structural analysis revealed that emulsified pectin (EP) gel microbeads were harder and more cohesive than those of emulsified alginate (EA). EP showed high swelling capacity and slower matrix degradation in aqueous media, suggesting that oleic acid is mainly located on the surface of EP microbeads. EA and EP matrices having p-nitrophenyl palmitate (C-16 ester) as tracer dissolved into oleic acid and in the presence of lipase confirmed this hypothesis which is consistent with EP better phage protective capability. Surface analysis of gel microbeads by scanning electron microscopy revealed strong differences between EP and EA gel microbeads. Phage release kinetics was tested using semi-empirical mathematical models. Experimental curve best fitted the Korsmeyer-Peppas model, predicting transport mechanisms according to the high swelling and degradation of EP. The proposed encapsulation model represents an innovative technology for phage therapy, which can be extrapolated to other therapeutic purposes, using a simple environmentally friendly synthesis procedure and cheap food-grade raw materials.

Tailoring doxorubicin sustainable release from biopolymeric smart matrix using congo red as molecular helper.

Doxorubicin (Dox) was co-encapsulated with congo red (CR) in order to increase drug encapsulation and sustain the release from gel microbeads composed of alginate-carboxy methyl guar gum (68/32) for oral controlled delivery. No release of either cargo molecule from the microbeads at pH 1.2 within 90 minutes was detected. However, 62% CR and 16% Dox were released from the gels at pH 7.4 at 37 °C in 8 hours when both the cargo molecules were studied alone. Presence of CR in the formulation reduces the release of Dox by about 25-30% under the same experimental conditions. Rheological properties of the formulations have been investigated at different temperatures between 20 and 37 °C. Shear thinning behavior was observed by steady-shear flow experiments for all formulations, and no yield stress was observed for any of the formulations. The temperature effect on Alg-CMGG-Dox-CR evidenced a synergic action between Dox and CR. Dynamic frequency sweep tests were performed to study the viscoelastic properties of the formulations. The patterns observed for Alg-CMGG indicated physical gel characteristics; however, all other formulations showed behaviour typical of concentrated solutions. These results confirm the interaction of Dox and CR, and the concomitant positive effect on sustainable release in oral delivery.

Copper ion adsorption by chitosan nanoparticles and alginate microparticles for water purification applications

Chitosan and alginate are natural carbohydrate materials used to remove heavy metal ions from aqueous solutions. In this research, chitosan gel nanoparticles were formed by ionic cross-linking with sodium tripolyphosphate and characterized by zetasizing and TEM. The equilibrium adsorption capability of chitosan nanoparticles and calcium-alginate gel microbeads for Cu2+ from copper sulfate solutions at fixed pH has been explored. Results show that the adsorption behavior of chitosan and alginate in the low concentration region follows the Langmuir isotherm. Approximately linearly increasing adsorption behavior was observed at higher solution concentrations. Chitosan gel nanoparticles exhibit a minor increase in adsorption capacity compared to other forms of chitosan. Alginate has significantly higher capacity than chitosan, which can be attributed to a comparatively higher density of adsorption sites. Combination particles consisting of alginate microbeads coated with chitosan nanoparticles possess an intermediate maximum adsorption capacity, corresponding to the weight ratio of the alginate and chitosan.

Hyper Alginate Gel Microbead Formation by Molecular Diffusion at the Hydrogel/Droplet Interface

We report a simple method for forming monodispersed, uniformly shaped gel microbeads with precisely controlled sizes. The basis of our method is the placement of monodispersed sodium alginate droplets, formed by a microfluidic device, on an agarose slab gel containing a high-osmotic-pressure gelation agent (CaCl(2) aq.): (1) the droplets are cross-linked (gelated) due to the diffusion of the gelation agent from the agarose slab gel to the sodium alginate droplets and (2) the droplets simultaneously shrink to a fraction of their original size (<100 μm in diameter) due to the diffusion of water molecules from the sodium alginate droplets to the agarose slab gel. We verified the mass transfer mechanism between the droplet and the agarose slab gel. This method circumvents the limitations of gel microbead formation, such as the need to prepare microchannels of various sizes, microchannel clogging, and the deformation of the produced gel microbeads.

Synthesis and application of vinylpyridine containing ion‐imprinted copolymer gel microbeads for Cu(II) solid‐phase extraction

The influence of polymer matrix on the extraction efficiency for Cu(II) and selectivity against metal ions such as Ni(II), Cd(II), Pb(II) of Cu(II) imprinted copolymer gels was described. The functional monomers investigated include the weakly basic 4-vinylpyridine (4-VP) and its mixure with the acidic and hydrogen binding methacrylic acid. Copolymer gels were prepared by dispersion cross-linking copolymerization using Cu(II)-4-(2-pyridylazo)resorcinol complex, Cu(II), or 4-(2-pyridylazo)resorcinol as templates. The chemical structure and morphology of the Cu(II)-imprinted microbeads are defined using elemental analysis, Fourier transform infrared spectroscopy, and scanning electron microscopy. Extraction efficiencies of newly synthesized sorbents were studied by batch procedure. The prepared copolymer gel with 4-VP as monomer and Cu(II)-4-(2-pyridylazo)resorcinol complex has higher capacity and selectivity toward Cu(II) than the copolymer gels prepared using the mixture of methacrylic acid and 4-VP. This new sorbent can be used as an effective SPE material for the highly selective preconcentration and separation of Cu(II) in sea water samples. It shows high mechanical and chemical stability.

Cytotoxicity evaluation of reactive metabolites using rat liver homogenate microsome-encapsulated alginate gel microbeads

We present an improved cytotoxicity test for reactive metabolites, in which the S9 microsomal fraction of rat liver homogenate is encapsulated in alginate gel microbeads to avoid cytotoxic effects of S9-self-generated toxicants, microsomal lipid peroxides. The S9-encapsulated gel microbeads were prepared by a coaxial two-fluid nozzle and surfaces of the microbeads were coated with poly-L-lysine (PLL). Although the initial metabolic rate of the S9-encapsulated gel microbeads was about 20% slower than that of bare S9, the microbeads prevented the leakage of microsomal lipid peroxides thanks to the dense alginate and PLL polymer networks. In fact, the half maximal effective concentration of the indirect mutagen cyclophosphamide on NIH3T3 cells in the presence of the S9-encapsulated gel microbeads was about 5 times higher than that in the presence of bare S9. Use of the S9-encapsulated gel microbeads enabled the more accurate evaluation of the cytotoxicity of the reactive metabolites without the S9-based cytotoxicity.

Survivability of probiotics encapsulated in alginate gel microbeads using a novel impinging aerosols method

Encapsulation of probiotic bacteria in cross-linked alginate beads is of major interest for improving the survivability in harsh acid and bile environment and also in food matrices. Alginate micro beads (10–40 μm) containing the probiotics Lactobacillus rhamnosus GG and Lactobacillus acidophilus NCFM were produced by a novel technique based on dual aerosols of alginate solution and CaCl 2 cross linking solution. Extruded macro beads (approximately 2 mm diameter) produced by the conventional method and micro beads produced by novel aerosols technique offered comparable protection to L. rhamnosus in high acid and bile environment. Chitosan coating of micro beads resulted in a significant increase in survival time of L. rhamnosus from 40 to 120 min in acid condition and the reduction in cell numbers was confined to 0.94 log over this time. Alginate macro beads are more effective than micro beads in protecting L. acidophilus against high acid and bile. Chitosan coating of micro beads resulted in similar protection to L. acidophilus in macro beads in acid and extended the survival time from 90 to at least 120 min. Viability of this organism in micro beads was 3.5 log after 120 min. The continuous processing capability and scale-up potential of the dual aerosol technique offers potential for an efficient encapsulation of probiotics in very small alginate micro beads below sensorial detection limits while still being able to confer effective protection in acid and bile environment.

Synthesis and Characterization of o-Hydroxybenzophenone Chromophore Bonded to Aminopropyl Silica Gel Microbeads

Abstract o-Hydroxybenzophenone (Hbp) chromophores were immobilized on 3-aminopropyl silica gel (SiO2–NH2) by a reaction of succinimidyl 4-(4-benzoyl-3-hydroxyphenoxy)butanoate (2a) and -undecanoate (2b) with SiO2–NH2 to produce microbeads 1a and 1b in 25 and 21% yield, respectively. The bond formation was confirmed by appearance of an absorption at 1550 cm−1 due to the N–H bending of the amide bond in the IR spectra of 1a and 1b. In order to enhance the thermal and photochemical stabilities, the 1a and 1b residual amino group was acetylated by Ac2O in CHCl3 to produce 7a and 7b. Acetylation occurred on the aminopropyl group. However, it did not occur on the phenolic OH group of Hbp from the IR spectra where the absorptions of the bending of the methyl group and the bending of the phenolic OH group appeared at 1375 and 1348 cm−1, respectively. By degradation experiments, it was found that thermal and photochemical stabilities were high in 7a and 7b compared with that of 1a and 1b.